Location: Sunflower and Plant Biology Research
Project Number: 3060-21000-043-23-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2019
End Date: Dec 31, 2020
Objective 1. Determine the nature of the interaction of between soil bacteria, Sclerotinia, and other fungi. Objective 2. Identify the distribution of the 42 bacterial and archaeal OTUs. Objective 3: Target the fungal component of the rhizosphere to determine if Arbuscular Mycorrhizal Fungi (AMF) are correlated with resistant plant genotypes.
1. To quantify negative interactions between any of the 42 bacterial and archaeal taxa of interest with S. sclerotiorum, individual microbes will be isolated into pure culture by suspending bulk soil from the Carrington field plots in tris-buffered phosphate (TBP). Soil suspensions will then be plated on a series of taxa-specific nutrient media using serial dilutions. Inoculated media will be incubated at 27°C for 10-30 days to collect potentially slow growing taxa. At multiple time points throughout the incubation, individual colony isolates will be collected, re-grown in a liquid culture of the media from which they were collected. Isolates will be identified using PCR amplification of the 16S rRNA locus, and cross referenced against the 42 OTUs sequences identified in the previous study. Isolates identified as members of the 42 OTUs will be tested for antagonism against Sclerotinia in growth inhibition assays on nutrient media agar. Microbial co-cultures will be incubated at 25°C for 15 days. Negative interactions will be scored by growth inhibition at the mycelial interface. Isolates that inhibit Sclerotinia growth in these in vitro assays will then be targeted for testing Sclerotinia inhibition in future greenhouse studies. 2. Using an existing database of microbes amplified from 237 soils sampled worldwide and across a range of ecological and agricultural settings (Delgado-Baquerizo et al. 2018), we will search for the global distribution of the 42 OTUs we identified in our previous study, and determine what soil characteristics are correlated with these putative Sclerotinia resistant microbes. Using the USDA-NRC soil survey, we will identify soils within the United States that should harbor these Sclerotinia-resistant soil bacteria and seek to sample those soils. We will then work with local land-grant universities and USDA-ARS research centers to sample soils that may harbor these potential bacterial taxa. Collected soils will be subjected to a variety of assays focused on identifying these Sclerotinia microbial taxa and assessing their ability to convey Sclerotinia resistance, including greenhouse studies of infection assays with resistant sunflower genotypes, 16S profiling to determine if these taxa are present in the soils, and bacterial isolation followed by in vitro Sclerotinia competition assays as mentioned in Objective 1. 3. We will use existing DNA isolations from our preliminary analysis and subject them to PCR amplification using the AMF-preferential primers, NS31 and AML2, then prepared and sequenced using Illumina MiSeq 2x150. Cleaned and filtered sequences will then be analyzed using the Qiime 2 software (Boylen et al. 2018) using the extensive and well curated AMF database MaarjAM (Opik et al. 2010). Presence or absence of AMF will be scored for all genotypes, and relative abundance of AMF sequences will be quantified and statistically analyzed to see if there is a correlation between resistance and any AMF OTUs identified. Putative AMF taxa could then be targeted for spore isolation for use in future greenhouse experiments inoculating sunflowers with potentially disease-suppressing AMF.